Detection of the presence of a specific DNA or RNA sequence in a sample is required for a variety of experimental, diagnostic and therapeutic purposes, e.g. detection of a specific mutation in a sample of amniotic fluid, parenterage testing, testing for incorporation of a viral DNA into a cell's genomic DNA, etc. The task of direct detection of a specific DNA or RNA sequence, which is routinely performed by the use of an appropriately labelled probe, is often hindered by the fact that the specific DNA or RNA is present in a sample only in minute amounts.
Examples of methods which enable the amplification of DNA sequences present in a sample in only minute quantities are: LCR (ligase chain reaction), 3SR (self-sustained sequence replication) or PCR (polymerase chain-reaction). In PCR a sample is contacted with a primer DNA complimentary to a 3' end sequence of the specific DNA, a DNA polymerase and with single DNA nucleotides. Following a number of replication cycles, the sample is enriched with the specific assayed DNA. A typical cycle of PCR comprises three distinct stages: a first stage in which the double-stranded DNA is melted to two single strands; a second stage of annealing of the primer to the single-stranded DNA; and a third stage of polymerization where the annealed primers are extended by the DNA polymerase, to produce a double-stranded DNA. The cycle of melting, annealing and DNA synthesis is repeated many times, the products of one cycle serving as templates for the next ad thus, each successive cycle enriches the sample with the specific DNA.
PCR suffers from several shortcomings, the most serious of which being its lack of specificity. The effective hybridization temperature, i.e. the temperature in which the two strands of DNA hybridize, determines the specificity of the reaction. A low effective hybridization temperature results in a higher percentage of non-specific binding. In PCR this temperature, which is defined by the temperature of the annealing stage, is relatively low and this brings about non-specific binding of the probe to the target sequences resulting in amplification of undesired sequences which brings about a relatively high background reading.
This non-specificity also requires an additional and time-consuming detection procedure such as electrophoretic separation of the amplification products on an agarose gel, in order to separate between the various amplification products, and does not enable detection of the presence of the assayed DNA by a mere detection of amplification.
PCR also suffers from a severe problem of contamination which is due to amplification of sequences that did not originate from the test sample being sequences unintentionally introduced to the sample.
Another disadvantage of PCR is that it is a complex procedure. Typically, each of the stages of melting, annealing and polymerization is carried out at a different temperature, e.g. melting at 94.degree. C., annealing at 50.degree. C. and polymerization at 72.degree. C. Since the samples have to be constantly cycled through several temperatures a special apparatus is required rendering the procedure laborious and time consuming.
Another shortcoming of PCR is in the time required therefor. A typical cycle lasts several minutes, and usually 25-30 cycles are required to produce sufficient copies of amplified DNA. Thus, a typical PCR even in a completely automated system lasts at least 2 to 3 hours.
Finally, PCR is basically suited for the detection of DNA sequences. Where detection of RNA sequences is desired, RNA has to be converted first to DNA (by reverse transcription). This conversion to DNA requires additional time, effort and enzymes, and also introduces many errors due to the inherent inaccuracy of reverse transcription.
It should be noted that although PCR is advantageous in obtaining large amounts of a specific DNA, such as for producing large quantities of probes for genetic assays, it is often an "over-kill" where merely the presence of a specific DNA sequence in a sample is to be assayed.
Other such methods such as 3SR (WO PCT 89/05631) and Target Nucleic Acid Amplification/Detection (WO PCT 89/05533) are relatively rapid isothermal processes for DNA detection. However, these methods also suffer from relatively effective low hybridization temperatures which are even lower than those of PCR, typically in the range of 37.degree.-41.degree. C. These low temperatures drastically reduce the specificity of the procedure due to non-specific probe-target binding, and in cases of clinical diagnostics, this may result in an intolerable level of misdiagnosis.
Additionally, amplification strategies such as Target Nucleic Acid Amplification/Detection that are based on the amplification properties of a replicase-type enzyme are unreliable due to the possibility of spontaneous RNA amplification in the absence of target (Chetverin-AB, et al., J. Mol. Biol., 222(1), 3-9 (1991)).
It is the object of the invention to provide a method for the detection of a nucleic acid sequence which is:
(i) reliable and sequence specific due to the minimalization of incorrect target-probe hybridization; PA1 (ii) relatively rapid; PA1 (iii) essentially isothermic eliminating the need for specialized and expensive apparatus; PA1 (iv) relatively simple, not requiring the addition of a large number of different enzymes or nucleotide pools; and PA1 (v) amenable to automation by enabling the amplification process itself to be indicative of the presence or absence of the nucleic acid sequence to be assayed. PA1 (a) reacting the sample with a detection ensemble comprising: PA1 (b) incubating under conditions to allow hybridization of said first DNA molecule and said second DNA molecule and were present also said third DNA molecule with said assayed nucleic acid sequence, and optionally adding a ligase to allow ligation of adjacent ends of said first, second and third DNA molecules; PA1 (c) adding transcription reagents comprising an RNA polymerase and RNA nucleotides and incubating under conditions to allow the formation of RNA transcripts having said triggering RNA sequence; PA1 (d) contacting the RNA transcripts with an RNA amplification ensemble in which the triggering RNA sequence induces formation of RNA molecules containing the signal RNA sequence; and PA1 (e) detecting the presence of said signal RNA sequence, positive results indicating the presence of said assayed nucleic acid sequence in said sample.
Further objects of the invention will become clear from the following description.